Complement C3 contributes to synaptic elimination during brain development and is elevated in Alzheimer's disease (AD);but whether C3 is involved in early synaptic loss in AD remains unknown. C3 and its receptor CR3 also mediate microglial uptake and degradation of amyloid-beta (Abeta), a key protein in AD. Thus, elevated C3 may contribute to both the removal of toxic Abeta species and the aberrant tagging of neuronal synapses for removal, resulting in synaptic loss in AD brain. C3 knockout (C3KO) are C3-deficient through life. Aged male C3KO mice have more synapses in hippocampus and cortex, neurons in CA3 of hippocampus, increased gliosis, and perform better in cognitive tests compared to WT mice. Because complement-mediated synaptic removal takes place during development, it is difficult to dissect the developmental versus aging effects of C3- deficiency. Therefore, we propose to generate the first complement C3 conditional knockout mouse models (C3 cKO) using inducible and constitutive Cre-loxP systems that to assess the CNS effects of global C3KO after brain development and cell-specific C3KO throughout life. Dr. Carroll, our collaborator, has generated chimeric floxed C3 mice and is breeding them for germ-line transmission (C3fl/fl) so that they can be crossed with various Cre-mouse lines to eliminate C3 in a cell type- and/or age-specific manner, generating a novel tool for researchers in many fields. We hypothesize that global complement C3-deletion after brain development and C3-deletion in myeloid cells through life will be protective against age-dependent synapse and neuron loss in hippocampus. Therefore, we will develop two C3 cKO mouse models and compare the effects of lifelong C3-deficiency (C3KO) with C3-deficiency after brain development or in myeloid cells (only) on synapses, neurons, and glia.
In Aim 1, we will generate floxed complement C3 mice that normally express C3 protein, C3-LacZ and C3-LacZ;ZP-3-Cre mice. Dr. Carroll has generated chimeric floxed C3 and C3-LacZ mice and is breeding each line for homozygous germline transmission. His lab will characterize these mice and cross the C3-LacZ mice with ZP-3-Cre mice to determine C3 expression in mice.
In Aim 2, we will generate C3 inducible conditional knockout mice to assess the effects of global C3 deletion initiated after brain development. We will generate C3fl/fl;UBC-Cre-ERT2+/- mice by crossing the C3fl/fl mice from Aim 1 to ubiquitin-promoter driven Cre-Estrogen receptor 2 mice, treat the mice with tamoxifen at P60, and examine synapses, neurons and glia in hippocampus at 4 mo and 12 mo of age.
In Aim 3, we will constitutively knockout C3 in myeloid cells to determine whether C3 produced by these immune cells contributes to synaptic pruning and neuronal health. We will generate C3fl/fl;LysMCre+/- mice and examine mice for changes in synapses, neurons and glia in hippocampus at P30, 4 mo and 12 mo of age. This study will provide important data determining the role of C3 on brain wiring, which will lead ultimately to future studies in models of neurodegenerative diseases.

Public Health Relevance

Complement C3 helps mediate synaptic elimination during the development of mouse brain and is upregulated in Alzheimer's disease, a condition in which synaptic loss is an early event. Our recent work shows that aged C3 knockout mice were protected against age- and brain region-dependent synapse and neuron loss, and had superior cognitive performance compared to age-matched normal mice. In order to distinguish the C3 effects on development versus aging, we propose to generate two C3 conditional knockout mouse models in which C3 expression can be shut off at any age by injection of a chemical or, deleted in specific immune cells (only) throughout life.